Daniele Ricci

Haemodynamic and ultrastructural observations on the rat liver after two-thirds partial hepatectomy*

Rat liver ultrastructure was investigated after partial hepatectomy (PH), by scanning and transmission electron microscopy. Portal pressure was monitored before and after PH and, after killing performed at 6, 12, 24, 48 h and 10 d, regenerating livers were fixed by portal vein perfusion under haemodynamic conditions identical to those existing in vivo. An early and persistent increase in portal pressure after PH was found (P<0.01 for normal vs sham‐operated controls). Ultrastructural study showed sinusoid dilatation and disappearance of the sieve‐plate arrangement of small endothelial pores, thus leaving the parenchymal liver cell surface directly exposed to portal blood. Widening of sinusoids, endothelial fenestrations, intercellular spaces and spaces of Disse, was accompanied by dilatation of bile canaliculi. At 10 d, liver ultrastructure had returned to normal. Our observations suggest that a rise in portal pressure, as a consequence of PH, may be related to the observed ultrastructural changes in the liver.

Identification of PI-PLC β1, γ1, and δ1 in rat liver: Subcellular distribution and relationship to inositol lipid nuclear signalling

Abstract The subcellular distribution of PI-PLC β1, γ1, and δ1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC β1 is predominantly nuclear, while γ1 is largely cytoplasmic and δ1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms β1 and γ1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-β1 and -γ1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC β1 and γ1 play a central role in modulating the nuclear phosphoinositide cycle.

Changes of Nuclear PI-PLC γ1 During Rat Liver Regeneration

Abstract We have previously demonstrated that rat liver nuclei contain PI-PLC β 1 and γ 1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669–678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP 2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (β 1 , γ 1 , δ 1 ) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC β 1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear γ 1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC δ 1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC γ 1 that paralleled the in situ observation whereas the β 1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP 2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP 2 , obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the γ 1 isoform of PI-PLC.

The effect of high temperature on blood glucose level in two teleost fish (Ictalurus melas and Ictalurus punctatus)

Black and channel catfish (Ictalurus melas and Ictalurus punctatus) were gradually acclimated to 36°C. Ictalurus punctatus tolerated the higher temperature better than Ictalurus melas. An intravenous glucose tolerance test was performed on Ictalurus punctatus by intravenously injecting 25 mg/100 g body weight of d-glucose. Both in the control and in acclimated fish, the blood glucose peak was reached after 1 min, after 5 min it began to decrease and after 4 hr it was drastically reduced, reaching the starting level after 24 hr. There was no significant difference between the two groups of animals until the 24th hour, where the glucose level in blood was significantly higher (P < 0.05) in acclimated fish than in control animals. Immuno-histochemical and electron microscopy studies indicated the presence of insulin-positive beta cells with characteristic cytoplasmatic electron-dense granulations, without any apparently histomorphological difference between acclimated and control fish.

Nuclear inositol lipids in Friend erythroleukemia cells. Changes related to differentiation induced by hexamethylenebisacetamide.

Subcellular distribution of inositol lipids has been studied in Friend Erythroleukemia Cells following induction to erythroid differentiation with hexamethylenebisacetamide, after labelling with [3H]myo-inositol. In situ autoradiography indicated that inositol-derived molecules were present also in the nuclear compartment of uninduced and induced cells. Fractionation studies showed that the nuclear polyphosphoinositides were deeply changed after short induction times, while the whole cell inositol lipids resulted only slightly modified by the inducer. The nuclear recovery of phosphatidylinositol 4,5-bisphosphate was largely increased after 2 hrs of induction, suggesting that inositol lipid metabolism is involved in the early differentiation events occurring at the nuclear level.

Morphological and functional evaluation of isolated rat hepatocytes in three dimensional culture systems.

Various three-dimensional configurations, such as polyester tissue and woven-nonwoven, hydrophilic polyester fabric, either collagen-coated or uncoated, were investigated as potential scaffold for hepatocyte culture, in view of their use in bioreactors for hybrid liver support systems. Attachment, morphology and ultrastructure of primary adult rat hepatocytes were evaluated, as well as urea production and ammonium detoxification during a 24h incubation period in serum-free tissue culture medium. As control, hepatocytes were also plated onto collagen-coated dextran microcarriers and on plastic petri dishes, either collagen-coated or uncoated. In all the three-dimensional cultures, hepatocytes appeared morphologically intact without any statistically significant difference in metabolic activity. Collagen-coating did not influence cell attachment to polyester substrates, whereas woven-nonwoven hydrophilic polyester fabric may offer some potential advantages as three-dimensional system for hepatocyte culture in hybrid liver support systems.

Argyrophil Cells in the Rat Duodenum after Gastrectomy

Two different techniques of reconstruction after total gastrectomy were performed in rats: Roux-en-Y (RY) and jejunal interposition (JI). Sham-operated rats were used as controls. Sixteen weeks after surgery the animals were sacrificed. The distribution of the enteroendocrine cells in the duodenal and proximal jejunal mucosa was examined using the Grimelius silver nitrate stain. The frequency of the Grimelius-positive cells was estimated as number of cells per square millimeter of gut surface and as number of cells per transverse section of intestine. Intestinal villi height was also measured in the same duodenojejunal tracts. After gastrectomy, the JI rats showed a better weight gain than RY rats (p less than 0.05). A significant difference in the distribution of intestinal argyrophil cells was seen. The bypassed duodenojejunal tracts in RY rats showed a marked reduction of the Grimelius-positive cells in comparison with JI rats and controls (p less than 0.01). A reduction in the number of argyrophil cells in the proximal duodenum was also noticed in the JI rats in comparison with controls (p less than 0.01). A decrease in the villi height resulted in all the gastrectomized rats (p less than 0.01), but a significant decrease was also noticed in RY rats in comparison with JI (p less than 0.01). Our experimental observations suggested that the exclusion of the duodenum from intestinal transit after gastrectomy caused atrophy of the gut mucosa and a marked reduction in the number of argyrophil enteroendocrine cells in the bypassed tracts.